Selenophosphate synthetase forms the energy-rich compound, selenophosphate, from ATP and selenide. An essential cysteine residue in the Escherichia coli enzyme is replaced by selenocysteine, threonine, or arginine in isoenzyme forms present in eukaryotes and other bacteria. Free selenide is inactive as substrate for some isoenzyme forms. Sulfur transferases that deliver active sulfane sulfur via enzyme persulfide intermediates also can serve as selenium delivery systems in vitro and, when Secys is supplied as Se donor, the coupled system effectively replaces free selenide. However, in vivo, where Se:S ratios are very low, a Se-specific delivery enzyme together with its Se substrate should be essential. To determine whether the unidentified 33 kDa selenium binding protein purified from Methanococcus vannielii is a Secys-specific lyase or the Se-binding subunit of xanthine dehydrogenase, purification of the latter was undertaken. Catalytically active enzyme was purified from Clostridium purinolyticum in the absence of DTT, and isolation of the selenium-binding subunit is in progress. Also, possible coenrichment of Secys lyase activity and 33 kDa [75Se]protein from M. vannielii extracts will be monitored. The thiroedoxin reductase gene from human placenta was modified for expression in E. coli. by inserting the E. coli fdhF SECIS stem-loop structure between the C-terminal glycine codon and the UAA stop. Either a calcineurin or a poly his vector was placed at the N-terminus to allow isolation via a calmodulin or a Ni column, respectively. Activities of wild type (TGA codon) and Cys mutant (TGT codon) enzymes expressed in E. coli were low but significant and typical flavin spectra were exhibited. C-terminal structures of the expressed enzyme are to be determined. - selenophosphate, selenide, Se-transfer protein, selenocysteine